Tagged: metastasis

Harnessing the Power of Nanobodies: Inhibiting Metastasis of 4T1-12B Breast Tumor Cells

September 12, 2024

In this study, researchers show that treatment of 4T1-12B mouse breast cancer cells with this nanobody inhibits V-ATPase-dependent acidification of the media and invasion of these cells in vitro.

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Researchers recently developed a nanobody directed against an extracellular epitope of the mouse V-ATPase c subunit. Zhen Li, Mohammed A. Alshagawi, Rebecca A. Oot, Mariam K. Alamoudi, Kevin Su, Wenhui Li, Michael P. Collins, Stephan Wilkens, and Michael Forgac from Tufts University School of Medicine; Tufts University; Dana Farber Cancer Institute, Harvard Medical School; University of Minnesota School of Medicine; Prince Sattam Bin Abdulaziz University; Korro Bio; SUNY Upstate Medical University; and Foghorn Therapeutics, suggest that plasma membrane V-ATPases represent a novel therapeutic target to limit breast cancer metastasis. The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to control the pH of intracellular compartments as well as to transport protons across the plasma membrane of various cell types, including cancer cells.

On August 14, 2024, their research paper was published in Oncotarget’s Volume 15, entitled, “A nanobody against the V-ATPase c subunit inhibits metastasis of 4T1-12B breast tumor cells to lung in mice.”

The Research

Breast cancer is one of the most diagnosed cancers, accounting for almost one-third (30%) of all new diagnoses in women in 2022. At the time of diagnosis, 20–30% of patients with early-stage breast cancer will go on to develop metastatic breast cancer. 6–10% of all patients with breast cancer have stage IV disease at time of diagnosis. It has been shown that V-ATPase plays an important role in promoting the invasiveness of many cancer cell types, including breast cancer cells.

This study demonstrated that inhibiting cell surface V-ATPases can effectively block tumor cell invasion. The findings indicate that anti-V-ATPase antibodies targeting an extracellular region of the V-ATPase can suppress activity on the surface of cancer cells, as well as inhibit both in vitro invasion and in vivo metastasis in a mouse model. This represents a promising advancement toward developing a new therapy to limit breast cancer metastasis.

Results

A camelid nanobody against the N-terminus of the mouse V-ATPase c subunit was prepared using phage display. The nanobody was dimerized through disulfide bonding to create a bivalent molecule. The purified nanobody was detected using Coomassie blue staining and Western blotting. The apparent molecular weight of the dimer on SDS-PAGE was around 45 kDa, slightly faster than the predicted weight of 56.8 kDa. The nanobody was tested for its ability to inhibit V-ATPase-dependent acidification in mouse 4T1-12B cells. The nanobody treatment resulted in a similar increase in extracellular pH as treatment with concanamycin, a known V-ATPase inhibitor.

Combining both the nanobody and concanamycin did not significantly enhance the effect. The nanobody effectively inhibited V-ATPase-dependent extracellular acidification without affecting cell viability. The anti-V-ATPase nanobody was tested for its ability to inhibit in vitro invasion of 4T1-12B cells. Treatment with the nanobody significantly inhibited invasion, like its inhibition of extracellular acidification. The nanobody effectively inhibits both extracellular acidification and in vitro invasion of 4T1-12B cells with similar affinity.

The administration of the anti-V-ATPase nanobody was tested to determine its effect on tumor growth and metastasis in mice. Different amounts of the nanobody were administered to mice without any adverse effects. The effect of nanobody administration on in vivo metastasis was then tested using 4T1-12B cells implanted in the mammary fat pad. However, no significant difference in tumor volumes was observed between the control and nanobody-treated groups at the end of the study. Treatment with the anti-V-ATPase nanobody resulted in a significant reduction in lung metastasis but had no effect on tumor growth or leg metastases. No significant metastasis was observed in other organs. In contrast, treatment with the anti-GFP nanobody did not reduce lung metastases.

Discussion

The researchers’ previous results demonstrated that selective inhibition of cell surface V-ATPases using an antibody or bafilomycin showed potential in inhibiting invasion of breast cancer cells. However, the use of antibodies against the native c subunit proved challenging due to its conservation and limited exposure. To overcome this, a nanobody against a native epitope of the c subunit was developed through in vitro screening. This nanobody successfully inhibited cell surface V-ATPase activity in mouse 4T1-12B breast cancer cells and showed a correlation between inhibition of invasion and extracellular acidification. In mice, the nanobody treatment significantly reduced lung metastases, but had no effect on tumor growth or leg metastasis.

The study suggests that different mechanisms may be involved in tumor cell invasion in different tissues. The potential side effects of inhibiting cell surface V-ATPases were also discussed, highlighting the limited presence of these pumps in certain cells and the potential benefits of inhibiting osteoclast function for breast cancer metastasis to bone.

Overall, the findings support the use of inhibitory nanobodies against cell surface V-ATPases as a potential therapeutic approach to inhibit breast cancer metastasis.

“These results provide support for the use of an inhibitory antibody directed against an extracellular epitope of the V-ATPase as a potential anti-metastatic therapeutic to inhibit breast cancer metastasis.”

Click here to read the full research paper in Oncotarget.

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Oncotarget is an open-access, peer-reviewed journal that has published primarily oncology-focused research papers since 2010. These papers are available to readers (at no cost and free of subscription barriers) in a continuous publishing format at Oncotarget.com.

Oncotarget is indexed and archived by PubMed/Medline, PubMed Central, Scopus, EMBASE, META (Chan Zuckerberg Initiative) (2018-2022), and Dimensions (Digital Science).

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How Osteopontin Stimulates Mitochondrial Biogenesis and Cancer Metastasis

January 11, 2024

In this new study, researchers investigated the role of Osteopontin splice variants in cancer metastasis.

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Mitochondrial biogenesis, the process of increasing the size and number of mitochondria within cells, plays a crucial role in cancer metastasis. Metastasizing cells exhibit a unique metabolism that differs from the well-known Warburg effect observed in primary tumors. While primary tumors primarily rely on glycolysis for energy production, metastatic cells rely on oxidative phosphorylation and ATP generation for short-term energy needs. However, over longer time frames, mitochondrial biogenesis becomes a prominent feature in the success of metastasis.

In a new study, researchers Gulimirerouzi Fnu and Georg F. Weber from the University of Cincinnati’s James L. Winkle College of Pharmacy investigate the connection between short-term oxidative metabolism and long-term mitochondrial biogenesis in cancer metastasis. They hypothesized that Osteopontin splice variants, specifically Osteopontin-c, stimulate an increase in mitochondrial size through the activation of specific signaling mechanisms. On December 1, 2023, their new research paper was published in Oncotarget, entitled, “Osteopontin induces mitochondrial biogenesis in deadherent cancer cells.”

“Over longer time frames, mitochondrial biogenesis becomes a pronounced feature and aids metastatic success. It has not been known whether or how these two phenomena are connected. We hypothesized that Osteopontin splice variants, which synergize to increase ATP levels in deadherent cells, also increase the mitochondrial mass via the same signaling mechanisms.”

The Role of Osteopontin Variants in Mitochondrial Biogenesis

Deadhesion, the process of detaching cancer cells from the extracellular matrix, is known to induce metabolic reprogramming and promote cancer cell survival in circulation. Osteopontin (OPN), a cytokine produced by cancer cells, has been implicated in tumor progression and the development of metastases. It mediates tumor cell survival and expansion under deadherent conditions, making it an ideal candidate for studying the mechanisms behind mitochondrial biogenesis. The authors of the research paper focused on two Osteopontin splice variants, Osteopontin-a and Osteopontin-c, and their effects on mitochondrial biogenesis.

Through their experiments with breast tumor cells, the authors found that both Osteopontin-a and Osteopontin-c contribute to mitochondrial biogenesis in deadherent cells. However, Osteopontin-c was more effective in stimulating an increase in mitochondrial size compared to Osteopontin-a. The authors also observed that the autocrine effects of Osteopontin variants are critical for the survival and anchorage-independence of disseminating malignant cells.

The Role of CD44v and SLC7A11 in Osteopontin Signaling

To further elucidate the mechanism behind Osteopontin-induced mitochondrial biogenesis, the authors investigated the receptors involved in Osteopontin signaling. They focused on CD44, a cell surface receptor known to interact with Osteopontin, and its variant CD44v. The authors found that Osteopontin-induced mitochondrial biogenesis is mediated via the binding of Osteopontin to CD44v.

Additionally, the authors discovered that the chloride-dependent cystine-glutamate transporter SLC7A11 plays a crucial role in Osteopontin signaling. The upregulation and co-ligation of SLC7A11, along with CD44v, leads to the activation of PGC-1, a known inducer of mitochondrial biogenesis. Surprisingly, the authors found that peroxide, an important intermediate in this signaling cascade, acts upstream of PGC-1 and is likely produced as a consequence of SLC7A11 recruitment and activation.

In Vivo Implications and Therapeutic Targets

To validate the relevance of their findings in clinical settings, the authors analyzed gene expression profiles in breast cancer metastases and metastases from other types of cancers. They identified the master regulator of mitochondrial biogenesis, PPARG, as well as its downstream effectors NRF1 and BACH1, to be upregulated in various metastases. These findings suggest that the Osteopontin-induced activation of PGC-1 and subsequent mitochondrial biogenesis may play a crucial role in cancer metastasis.

The authors also conducted in vivo experiments using mouse models. They observed that suppression of the biogenesis-inducing mechanisms led to a reduction in disseminated tumor mass. These findings not only confirm the functional connection between short-term oxidative metabolism and long-term mitochondrial biogenesis in cancer metastasis but also provide potential mechanisms and targets for treating cancer metastasis.

Conclusion

This study provides valuable insights into the role of Osteopontin splice variants in regulating mitochondrial biogenesis in metastatic cancer cells. The researchers demonstrated that Osteopontin-c stimulates an increase in mitochondrial size through the activation of specific signaling mechanisms involving CD44v and SLC7A11. These findings have significant implications for understanding the metabolic adaptations of metastatic cancer cells and suggest potential targets for therapeutic interventions. Further research is needed to fully elucidate the intricate signaling pathways involved in Osteopontin-induced mitochondrial biogenesis and to explore the clinical applications of these findings in cancer treatment.

“This study confirms a functional connection between the short-term oxidative metabolism and the longer-term mitochondrial biogenesis in cancer metastasis – both are induced by Osteopontin-c. The results imply possible mechanisms and targets for treating cancer metastasis.”

Click here to read the full research paper in Oncotarget.

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Immunotherapy Response in Primary vs Metastatic Pancreatic Cancer

November 16, 2023

In this editorial, researchers delve into the immunotherapeutic challenges posed by the tumor microenvironment and liver metastasis in pancreatic cancer.

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Pancreatic ductal adenocarcinoma (PDA), a common type of pancreatic cancer, has proven to be largely resistant to immunotherapy, a treatment that uses the body’s immune system to fight cancer. Despite numerous successful pre-clinical trials using sophisticated PDA mouse models, clinical trials have failed to show a significant improvement in survival.

In a recent editorial, researchers Brian Diskin, Sarah Schwartz and George Miller from Trinity Health of New England shed light on the complex interplay between the immune system and pancreatic cancer. Their paper was published in Oncotarget on April 24, 2023, and entitled, “The critical immune basis for differential responses to immunotherapy in primary versus metastatic pancreatic cancer.”

Tumor Microenvironment and Liver Metastasis: Challenges in Pancreatic Cancer

The authors attribute PDA immunotherapy resistance to the unique characteristics of the tumor microenvironment (TME). The TME is often hypoxic and fibrotic, making it inaccessible to immune cells. Furthermore, the immune cells that do infiltrate the TME often have tolerogenic features, meaning they are more likely to tolerate the presence of cancer cells rather than attack them.

PDA most commonly metastasizes to the liver, an organ known for its immune tolerance. The liver is home to a diverse array of innate immune populations, including NK cells, Kupfer cells, NKT cells, and double negative T cells. Despite this, the liver is the most common location for metastasis from gastrointestinal cancers.

“It is an unfortunate fact that all failed clinical trials assessing immunotherapeutic efficacy were conducted in metastatic PDA, whereas basic preclinical investigations are usually performed in primary PDA using genetically engineered mouse models. We postulated that this dichotomy may explain the gap between preclinical promise and ultimate clinical failure.”

Divergent Responses to Immunotherapy: Primary vs. Metastatic

“The potentially divergent responses to immunotherapy in the respective environments of primary versus metastatic PDA within the same host has not been well-studied.”

The authors highlight the lack of research into the potentially divergent responses to immunotherapy in primary versus metastatic PDA. They argue that this gap in knowledge may explain the discrepancy between the promising results of pre-clinical trials and the disappointing outcomes of clinical trials.

In their research, they discovered that the TMEs of primary PDA and liver metastases differ significantly, and this difference plays a critical role in the site-specific response to immunotherapy. They found that liver metastases are uniquely resistant to immunotherapies, in stark contrast to the immunotherapeutic responsiveness of primary PDA.

“We discovered that the respective TMEs of primary PDA and liver metastases differ markedly and this fact plays a critical role in dictating site-specific PDA response to immunotherapy [6].”

The Role of B Cells

The researchers identified B cells as a key player in this differential response. They found that B cells constituted approximately 25% of the tumor-infiltrating lymphocytes in metastatic PDA liver deposits, compared to approximately 10% in primary PDA. They also discovered a novel population of CD24+CD44–CD40– B cells in the metastatic liver, which is recruited to the metastatic milieu by Muc1hiIL18hi tumor cells.

“[…] by targeting B cells or blocking CD200/BTLA, we demonstrated enhanced macrophage and T-cell immunogenicity, which enabled immunotherapeutic efficacy of liver metastases.”

However, the authors note that primary PDA sites lack this b-cell population. Instead, they are characterized by macrophages and effector T cells that have a higher ability to provoke an immune response. This makes their immunotherapeutic responsiveness far more robust than metastatic liver PDA.

Conclusion

This research underscores the importance of understanding the immune basis of differential responses to immunotherapy in primary versus metastatic pancreatic cancer. It highlights the need for further research into the role of the TME and immune cells like B cells in the response to immunotherapy. Such insights could pave the way for more effective treatments for this challenging disease.

“[…] our data suggest that models of primary PDA are poor surrogates for evaluating immunity or treatment response in advanced disease.”

Click here to read the full editorial paper in Oncotarget.

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The Evolution of Metastatic Cancer: Mechanisms and Drivers

April 14, 2023

In a new editorial, researchers explore genomic evolution in metastatic cancer, how therapy can drive it and the implications for developing new treatments.

The Evolution of Metastatic Cancer: Mechanisms and Drivers

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There are several theories that attempt to explain the genesis of cancer. One prominent theory is the genetic theory—proposing that cancer may arise from the accumulation of genetic mutations that alter the normal functioning of cells. These mutations can drive the formation of tumors, which can then spread to other parts of the body in a process known as metastasis. Metastatic cancer is often difficult to treat because it has evolved to become resistant to standard therapies.

“It is generally accepted that development of cancer is a slow process, likely spanning decades during which the developing neoplastic cells sequentially acquire genomic alterations that will eventually give rise to the primary tumor [1].”

In a new editorial, researchers Ditte S. Christensen and Nicolai J. Birkbak from Aarhus University discuss mechanisms of genomic evolution in metastatic cancer, how therapy can drive it and the implications for developing new treatments. Their editorial paper was published in Oncotarget on March 21, 2023, entitled, “Therapy drives genomic evolution in metastatic cancer.”

Therapy Can Drive Metastatic Cancer

Cancer cells are master adaptors and have a remarkable ability to evolve, especially in response to therapy. When cancer cells are exposed to chemotherapy, radiation or targeted therapies, they can develop resistance to these treatments by acquiring new genetic mutations. This can occur through a variety of mechanisms, including mutations in the genes that regulate cell division and DNA repair, as well as the acquisition of new genes that confer resistance to specific drugs.

In this editorial, the authors discuss how this process of genomic evolution can lead to the development of metastatic cancer. As cancer cells acquire new mutations that allow them to survive and grow in the presence of therapy, they may also acquire mutations that allow them to invade and colonize new tissues. This can lead to the development of new tumors in distant parts of the body, which are often more difficult to treat than the original tumor.

“How the ability to perform these multiple independent steps is acquired by cancer cells remains a mystery.”

Mechanisms of Metastatic Cancer

Understanding the mechanisms by which cancer cells evolve in response to therapy is essential for developing new treatments for metastatic cancer. The clonal bottleneck hypothesis and the gatekeeper mutation hypothesis are two different hypotheses that attempt to explain how cancer cells acquire the ability to metastasize and spread to distant parts of the body. The clonal bottleneck hypothesis proposes that metastatic cancer is the result of a single subclone of cancer cells from the primary tumor that successfully seeds new sites. According to this hypothesis, the cancer cells undergo a clonal bottleneck event where only a small number of cells from the primary tumor are able to survive and successfully colonize new tissues. This hypothesis suggests that the ability to metastasize is an inherent property of the subclone that successfully colonizes new sites.

On the other hand, the gatekeeper mutation hypothesis proposes that metastatic cancer is the result of a specific genetic mutation or mutations that act as gatekeepers, allowing cancer cells to metastasize and spread to new sites. According to this hypothesis, the ability to metastasize is acquired through the acquisition of one or more specific genetic mutations that allow cancer cells to bypass the normal checks and balances that prevent uncontrolled growth and invasion of surrounding tissues.

Exploring Gatekeeper Genomic Events

In a 2022 study, the authors of this editorial and their team explored the concept of gatekeeper genomic events by comparing primary and metastatic tumors on a large scale. Their large-scale analysis of more than 40,000 individual tumors from the AACR Genomics Evidence Neoplasia Information Exchange (GENIE) project found an increase in mutation burden and chromosomal instability in metastatic tumors, but no evidence of individual mutations driving the metastatic process itself. The concept of gatekeeper mutations remains a hypothesis, and further research is needed to explore this idea in more detail.

This study and others suggest that metastatic cancer dissemination involves a bottleneck event where a highly fit clone from a primary tumor successfully seeds distant sites. Strong selective pressure from anti-cancer therapy drives the acquisition of private driver mutations associated with therapy resistance in individual metastatic tumors. There is limited evidence for the existence of specific gatekeeper mutations. It is also possible that the primary driver of metastatic cancer is found outside the cancer cells.

“Indeed, it may be that a primary driver of metastatic cancer is to be found outside the cancer cells themselves, potentially through inflammation in the tumor-immune microenvironment or through interaction with a declining host immune system which may enable immune escape and sudden systemic dissemination by a highly proliferative primary tumor clone.”

Conclusions

In conclusion, the genetic theory of cancer proposes that cancer arises from genetic mutations that alter the normal functioning of cells, leading to the formation of tumors and metastasis. This editorial by Christensen and Birkbak highlights the process of genomic evolution in metastatic cancer and its implications for cancer treatment. Understanding the mechanisms by which cancer cells evolve in response to therapy is crucial for developing new treatments for metastatic cancer. Recent studies have shed light on the clonal origin of metastatic tumors and the role of selective pressure from anti-cancer therapy in the acquisition of private driver mutations associated with therapy resistance. While the concept of gatekeeper mutations remains a hypothesis, it is clear that the acquisition of aggressive cancer traits is the primary driver of metastatic potential. Further research is needed to explore this idea in more detail and develop effective therapies for metastatic cancer.

“It will be exciting to further explore these questions as more data becomes available on metastatic cancers, particularly with paired primary and metastatic tumor samples with sequential biopsies to facilitate the analysis of dynamic tumor evolution over time, rather than through static snapshots provided by samples obtained at a single time point.”

Click here to read the full editorial published in Oncotarget.

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Oncotarget is an open-access, peer-reviewed journal that has published primarily oncology-focused research papers since 2010. These papers are available to readers (at no cost and free of subscription barriers) in a continuous publishing format at Oncotarget.com. Oncotarget is indexed/archived on MEDLINE / PMC / PubMed.

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